DNA Methylation Status Of IL-17 Signaling Pathway Involvement In Articular Cartilage Inflammation Of Osteoarthritis

Osteoarthritis is a worldwide public health problem,with an incidence of about 40%over the age of 70 years in the world.The disease not only causes pain,joint deformity and dysfunction,but also manifestations of metabolic syndrome.OA is not only a disease of degeneration,but also an inflammatory disease.The loss of articular cartilage caused by chronic inflammation is the key pathological change of OA.IL-17 is an important pleiotropic cytokine,which can induce the release of other cytokines,increase neutrophil chemotaxis,stimulate synoviocytes inducing nitric oxide synthase and MMPs and activate cartilage degradation enzymes.IL-17 involves in inflammation and destruction of articular cartilage.Recent reports show that expressions of IL-17 in synovial fluid and synovium in OA increased and associated with the severity of disease.Meanwhile,the level of IL17R in chondrocytes also increased.These evidences suggest that IL-17 and its receptor are involved in articular inflammation.Studies have shown that IL-17 plays its biological effects through the signaling pathway:IL-17 binding to the receptors IL17RA and IL17RC on target cell membrane,the intracellular molecules ACT1 and TRAF6 transducting the signals,and finally activating NF-?B.However,the expression and activation of the IL-17 signaling pathway in articular cartilage of OA is not clear.Epigenetic regulation plays an important role in the pathogenesis of OA,and DNA methylation is closely related to inflammatory diseases.Whole genome methylation studies of OA have identified demethylated CpG sites or regions that are enriched in genes related to immunity or inflammation.Targeting DNA methylation analysis showed that differential methylation status of specific sites in gene promoter or enhancer such as TNF?,IL-1?,IL-6,IL-8 and leptin related to the increased expression of these genes in articular cartilage or chondrocytes in OA.Studies have shown that IL17RC expression is enhanced in elderly patients with macular degeneration,which is regulated by promoter hypomethylation.However,the role of DNA methylation in the expression and activity of IL-17 signaling pathway in OA cartilage has not been reported.[Objectives]1.To investigate the expressions of key proteins in IL-17 signaling pathway and the activity of NF-?B pathway in the articular cartilage of OA,and to explore the role of IL-17 signaling imbalance in OA cartilage inflammation.2.To evaluate the DNA methylation status of key genes of IL-17 signaling pathway in articular cartilage of OA,and to analyze the regulatory effect on gene expression.3.To investigate the effect of 5-Aza-dC on methylation status and gene expression of IL-17 signaling pathway in vitro,and to analyze the impact of 5-Aza-dC on chondrocytes inducing cytokines and chemokines.[Methods]1.The cartilage samples were collected from knee OA donors.The degraded and fibrous cartilage as the experimental group,the fully intact cartilage with little fibrillation and no exposed bone as control group.IL-17 level of the synovial fluid was detected by ELISA.Expression and activity of IL-17 signaling pathway of articular cartilage were detected by real-time PCR,western blotting and immunohistochemistry.By proteome profiler array,we further observed the relative levels of 41 proteins and 4 serine or tyrosine phosphorylation sites involved in NF-?B signal transduction.2.To investigate the DNA methylation status of IL-17 signaling pathway,9 CpG islands near transcription start site(TSS)of IL17RA,IL17RC,ACT1,TRAF6 and RELA genes were quantitatively measured by Methyl Target-seq technology,which combined sodium bisulfite treatment with second-generation sequencing.A total of 256 CpG loci were anaylzed on Illumina Miseq.Further analysis was performed to evalute the regulation effect of DNA methylation on gene expression.3.Isolation,culture and identification of chondrocytes from OA cartilage were performed.In the culture of chondrocytes in vitro,demethylation drugs 5-Aza-dC was used to administrate chondrocytes.By MethylTarget-seq,changes of DNA methylation status of genes in IL-17 signaling pathway were evaluated,real-time PCR were performed to detect gene expression levels of mRNA.By cytometric bead array,cytokines and chemokines in the supernatant of cultured chondrocytes were measured.[Results]1.The role of IL-17 signaling pathway on chronic inflammation of OA cartilage(1)First,the level of IL-17 in synovial fluid of patients with OA(n=20)and the control group of patients with joint injury(n=20)were detected.ELISA results showed that IL-17A level in synovial fluid of OA patients was significantly higher than that in control group.IL-17 level in synovial fluid was observed be higher than in serum in OA patients.It is suggested that IL-17 is over expressed and the inflammation is more localized in the joint of OA patients.(2)Then,the degraded and fibrous cartilage as the experimental group(n=26),the fully intact cartilage with little fibrillation and no exposed bone as control group(n=26),we detected the expressions of transmembrane receptor(IL17RA,IL7RC),signal transduction molecules(ACTT1,TRAF6)and nuclear factor NF-?B(RELA/p65).Increased mRNA expressions of IL17RA,ACT1,TRAF6 and RELA/p65 were found in the degraded and fibrous cartilage by real-time PCR.Immunohistochemistry and western blotting showed that the proteins of IL17RA,IL17RC,ACT1 and RELA/p65 in affected cartilage of knee OA were significantly higher than that in non-affected areas.(3)Next,we further observed the expression and activity of NF-?B inflammatory pathway in cartilage of OA.The results showed that the relative levels of 41 proteins and 4 serine or tyrosine phosphorylation sites involved in NF-?B signal transduction in the lesions of cartilage were increased.Among them,four proteins(LTBR/TNFRSF3,IL-1RI,IKK1/IKK alpha/CHUK,BCL10)increased significantly compared with control group.It suggests that the NF-?B signaling pathway in OA cartilage is over expressed and activated.2.Role of DNA methylation status of IL-17 signaling pathway in cartilage inflammation of OA(1)First,the CpG island of key genes involved IL-17 inflammatory pathway was predicted with setting conditions as follows:Observed/Expected ratio>0.60,Percent C+Percent G>50.00%,Length>200 bp.CpG island located in the transcription start site(TSS)2K to the first exon of 1K downstream region.According to the above conditions,9 CpG islands were selected with a total of 256 CpG sites.(2)Next,the degraded and fibrous cartilage as the experimental group(n=40),the fully intact cartilage with little fibrillation and no exposed bone as control group(n=40).MethylTarget-seq technology,which combined sodium bisulfite treatment with second-generation sequencing,was performed to analyze the DNA methylation status on Illumina Miseq platform.DNA methylation profiles of single base were obtained.In the 256 CpG sites,26 differentially methylated CpG sites(DMSs)were found.Among these DMSs,21 CpG sites were significantly demethylated in affected cartilage in contrast to non-afFected cartilage,while the percentage methylation of the other 5 CpG sites was higher.ACT1 as the signal transduction molecule in the IL-17 signaling pathway is a differentially methylated gene.The promoter region of ACT 1 was significantly demethylated in affected cartilage.A significantly negative correlation was observed between TRAF6 gene expression and the percentage methylation of the CpG site located at +383 bp in OA cartilage.A significantly reciprocal trend between the percentage methylation of ACT 1 promoter and ACT 1 mRNA expression was observed.These results suggest that the methylation status of key genes involved in IL-17 signaling pathway may regulate the expression of these genes,which may contribute to the pathogenesis of OA.3.Effects of 5-Aza-2'-deoxycytidine on methylation status and gene expression of IL-17 pathway genes in OA chondrocytes(1)First,the chondrocytes were isolated by enzymatic digestion and cultured in vitro.Stained by toluidine blue and type ? collagen immunohistochemistry,the phenotype of chondrocytes was identified.The results show that we could obtain human articular chondrocytes with high purity.(2)Then,in order to simulate the local inflammatory environment of OAjoint,IL-17A of different concentrations(10ng/ml,50ng/ml)were used to stimulate chondrocytes in vitro.CBA showed that cytokines and chemokines produced by chondrocytes were enhanced,cytokines of IFNa,IFNy,MCP-1,IL-6,IL-12p70,IL-18,IL-23,IL-33 and chemokines of CXCL9,CXCL5,CCL20,CXCL1 in the supernatant were significantly increased compared with untreated group.(3)Finally,we used 5-Aza-dC to intervent the monolayer cultured chondrocytes,to observe the effects of 5-Aza-dC on methylation and gene expression of IL-17 signal pathway.MethylTarget-seq results showed that,the methylation status of IL17RA,IL17RC and TRAF6 were changed by 5-Aza-dC in 3d,methylation status of IL17RA and RELA were changed in 5d.Real-time PCR showed that the mRNA expression of IL17RC and ACT1 were enhanced significantly after 5-Aza-dC treatment.CBA results showed that cytokines and chemokines in culture supernatants secreted by the chondrocytes were significantly lower than those in control.[Conclusion]1.IL-17 level of synovial fluid in OA was increased.Key proteins of IL-17 signaling pathway(IL17RA,IL17RC,ACT1,TRAF6 and RELA)was over expressed and activated in degraded and fibrous cartilage,which may induce inflammation of cartilage.2.The key genes of IL-17 signaling pathway in OA cartilage showed lower methylation status,which increased the mRNA expression of related genes.DNA methylation modificaton may regulate the chronic inflammation of OA.3.5-Aza-dC could change the DNA methylation status and gene expression of IL-17 signaling pathway in chondrocytes,which may alter the inflammatory state of chondrocytes.[Significance]The demethylation of the key genes could regulate the expression and activity of IL-17 signaling pathway in OA cartilage,which is crucial to the chronic inflammation of cartilage.These results may contribute to elucidate the pathogenesis of this disease,and DNA methylation may be a new target for the treatment of OA.